Investigation of α,ω-Disubstituted Polyamine-Cholic Acid Conjugates Identifies Hyodeoxycholic and Chenodeoxycholic Scaffolds as Non-Toxic, Potent Antimicrobials

With the increased incidence of antibiotic resistance, the discovery and development of new antibacterials is of increasing importance and urgency. The report of the natural product antibiotic squalamine in 1993 has stimulated a lot of interest in the study of structurally simplified cholic acid-polyamine derivatives. We report the synthesis of a focused set of deoxycholic acid-polyamine conjugates and the identification of hyodeoxycholic acid derivatives as being potently active towards S. aureus MRSA and some fungal strains, but with no attendant cytotoxicity or hemolytic properties. Analogue 7e exhibited bactericidal activity towards a range of Gram-positive bacteria, while preliminary investigation of its mechanism of action ruled out the bacterial membrane as being a primary cellular target as determined using an ATP-release bioluminescence assay.


Introduction
Natural products have a proven track record of being an excellent source of novel antibiotics or molecules that provide inspiration for the development of new therapeutics [1,2]. Squalamine (1) is an example of a natural product antibacterial drug lead, originally isolated from tissues of the dogfish shark Squalus acanthias ( Figure 1) [3,4]. The unusual aminosterol exhibits broad-spectrum activity towards Gram-positive and Gram-negative bacteria as well as fungi and protozoa. The presence of both water-soluble groups (spermine and sulfate) and a lipophilic sterol core suggest that squalamine acts as a cationic amphiphilic antimicrobial targeting bacterial membranes, disruption of which leads to bacteria cell death [5].

Introduction
Natural products have a proven track record of being an excellent source of novel antibiotics or molecules that provide inspiration for the development of new therapeutics [1,2]. Squalamine (1) is an example of a natural product antibacterial drug lead, originally isolated from tissues of the dogfish shark Squalus acanthias ( Figure 1) [3,4]. The unusual aminosterol exhibits broad-spectrum activity towards Gram-positive and Gram-negative bacteria as well as fungi and protozoa. The presence of both water-soluble groups (spermine and sulfate) and a lipophilic sterol core suggest that squalamine acts as a cationic amphiphilic antimicrobial targeting bacterial membranes, disruption of which leads to bacteria cell death [5]. Closer examination of the interaction of squalamine with eukaryotic and prokaryotic membranes identified the natural product to be more selective for binding to the latter and that interaction with LPS-containing membranes was calcium ion dependent [5,6].
Squalamine induced a rapid release of intracellular ATP from Gram-positive bacteria and led to disruption of the bacterial membrane as observed in TEM images [5,7]. Weak hemolytic activities have been reported for squalamine, with an EC 50 of 80 µM for hemoglobin release from erythrocytes [8] and an EC 50 51 µM for propidium iodide entry into B lymphoma Wehi-231 cells [6], identifying it as a selective antibiotic lead for future development. In addition to demonstrating intrinsic antimicrobial properties, squalamine is also able to enhance the action of legacy antibiotics against Gram-negative bacteria [9]. In addition to membrane disrupting properties, a recent report has identified squalamine and structurally related mimics as inhibitors of the glycosyltransferase activity of Escherichia coli penicillin-binding protein PBP1b [10].
The initial report of the structure and biological activities of squalamine has stimulated widespread interest in the discovery of mimics that are structurally simplified and more easily prepared, as recently reviewed [11,12]. Many studies of squalamine mimics have used bile acids e.g., cholic acid (CA) (2), as the steroidal scaffold, due to their plentiful supply and the availability of a number of related structures e.g., (7)-deoxycholic acid, and isomers hyodeoxycholic acid (HDCA) (3), ursodeoxycholic acid (UDCA) (4) and chenodeoxycholic acid (CDCA) (5) (Figure 2). Closer examination of the interaction of squalamine with eukaryotic and prokaryotic membranes identified the natural product to be more selective for binding to the latter and that interaction with LPS-containing membranes was calcium ion dependent [5,6]. Squalamine induced a rapid release of intracellular ATP from Gram-positive bacteria and led to disruption of the bacterial membrane as observed in TEM images [5,7]. Weak hemolytic activities have been reported for squalamine, with an EC50 of 80 μM for hemoglobin release from erythrocytes [8] and an EC50 51 μM for propidium iodide entry into B lymphoma Wehi-231 cells [6], identifying it as a selective antibiotic lead for future development. In addition to demonstrating intrinsic antimicrobial properties, squalamine is also able to enhance the action of legacy antibiotics against Gram-negative bacteria [9]. In addition to membrane disrupting properties, a recent report has identified squalamine and structurally related mimics as inhibitors of the glycosyltransferase activity of Escherichia coli penicillin-binding protein PBP1b [10].
The initial report of the structure and biological activities of squalamine has stimulated widespread interest in the discovery of mimics that are structurally simplified and more easily prepared, as recently reviewed [11,12]. Many studies of squalamine mimics have used bile acids e.g., cholic acid (CA) (2), as the steroidal scaffold, due to their plentiful supply and the availability of a number of related structures e.g., (7)-deoxycholic acid, and isomers hyodeoxycholic acid (HDCA) (3), ursodeoxycholic acid (UDCA) (4) and chenodeoxycholic acid (CDCA) (5) (Figure 2). The particular position and/or stereochemistry of hydroxyl substitution on the cholic acid imparts 'facial amphiphilicity' [13][14][15][16], an attribute that has been exploited to develop bile acid-amine conjugates that exhibit wide ranging biological activities including acting as synthetic ionophores [17,18], plasmid transfection reagents [16,19], and antimicrobials [20][21][22][23][24]. In the case of the latter activity, cholic acid-amine conjugates have been found to exhibit strong to potent activity against Gram-positive and Gram-negative bacteria and fungi, with mechanisms of action attributed to bacterial membrane damage and/or membrane depolarization [12,14,20]. The mechanism of membrane disruption has been attributed to the observed ability of some mimics to exhibit antibiotic enhancing properties [22,23]. Any future exploitation of these bioactive molecules will require selectivity towards bacterial versus mammalian membranes-there are unfortunately several reports of cytotoxicity and/or hemolytic activities exhibited by squalamine mimics [13,15,20,23]. The particular position and/or stereochemistry of hydroxyl substitution on the cholic acid imparts 'facial amphiphilicity' [13][14][15][16], an attribute that has been exploited to develop bile acid-amine conjugates that exhibit wide ranging biological activities including acting as synthetic ionophores [17,18], plasmid transfection reagents [16,19], and antimicrobials [20][21][22][23][24]. In the case of the latter activity, cholic acid-amine conjugates have been found to exhibit strong to potent activity against Gram-positive and Gram-negative bacteria and fungi, with mechanisms of action attributed to bacterial membrane damage and/or membrane depolarization [12,14,20]. The mechanism of membrane disruption has been attributed to the observed ability of some mimics to exhibit antibiotic enhancing properties [22,23]. Any future exploitation of these bioactive molecules will require selectivity towards bacterial versus mammalian membranes-there are unfortunately several reports of cytotoxicity and/or hemolytic activities exhibited by squalamine mimics [13,15,20,23].
With only limited examples reported in the literature of C-24 amide-linked cholic acid-polyamine conjugates as squalamine mimics, we undertook a study to explore the effect of variation in the cholic acid head group, using hyodeoxycholic (3), ursodeoxycholic (4) and chenodeoxycholic (5) acids (Figure 2), and variation in polyamine chain length on antimicrobial properties. Compound cytotoxicity and hemolytic properties were also evaluated. Herein, we report on the synthesis and biological evaluation of this set of cholic acid-polyamine conjugates and the results of a preliminary mechanism of action evaluation.

Synthesis of Cholic Acid-Polyamine Conjugates
The target set of analogues required the synthesis of Boc-protected polyamine scaffolds 6a-f (Figure 3), which were prepared according to literature procedures [27][28][29][30]. The polyamines chosen covered a range of overall lengths, from spermine (polyamine PA-3-4-3) through to the longer chain length PA-3-12-3 variant. The set was chosen to allow exploration of the effect of chain length, lipophilicity, and positioning of positive charges on antimicrobial and cytotoxicity/hemolytic properties.
With only limited examples reported in the literature of C-24 amide-linked cholic acid-polyamine conjugates as squalamine mimics, we undertook a study to explore the effect of variation in the cholic acid head group, using hyodeoxycholic (3), ursodeoxycholic (4) and chenodeoxycholic (5) acids (Figure 2), and variation in polyamine chain length on antimicrobial properties. Compound cytotoxicity and hemolytic properties were also evaluated. Herein, we report on the synthesis and biological evaluation of this set of cholic acid-polyamine conjugates and the results of a preliminary mechanism of action evaluation.

Antimicrobial Activities
The antimicrobial activities of compounds 7a-f, 8a-f and 9a were determined against a panel of Gram-positive (methicillin-resistant Staphylococcus aureus (MRSA)) and Gramnegative (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii) bacteria and two fungal strains (Candida albicans and Cryptococcus neoformans) ( Table 1). The overall trend observed for the compounds was growth inhibition of the Gram-positive bacteria MRSA and the fungal pathogens and limited to no activity towards the Gram-negative bacteria. The hyodeoxycholic acid (HDCA) analogues 7a-f exhibited good to excellent activities against MRSA (MIC ≤ 0.19 to 3-4 µM) and C. neoformans (MIC ≤ 0.19 to 0.8 µM) and with the long polyamine chain variants 7e and 7f also exhibiting excellent activity against C. albicans (MIC ≤ 0.20 µM). In contrast, the ursodeoxycholic acid (UDCA) analogues 8a-f, while exhibiting similar MRSA levels of activity to the HDCA series, were typically less active as antifungals. The notable exception was the longest polyamine chain variant, 8f, which was identified as potently active against both MRSA and C. neoformans (MIC ≤ 0.19 µM). The single example of a chenodeoxycholic acid-polyamine conjugate (9a) exhibited a slightly different spectrum of antimicrobial activity compared to the other corresponding spermine derivatives 7a and 8a, with potent growth inhibition observed against MRSA and both fungal pathogens (MIC ≤ 0.21 µM) and was the only analogue tested that exhibited some degree of growth inhibition against a Gram-negative bacterium (MIC 6.8 µM against E. coli). What is notable in these results is the variation of activity, and the spectrum of activity against different microorganisms between these sets of cholic acid analogues, arising from differences in the position and stereochemistry of hydroxyl group substitution at C-6 or C-7. In the same biological assays, squalamine exhibited strong growth inhibition of the Gram-positive bacteria MRSA and Gram-negative organisms E. coli, K. pneumoniae and A. baumannii (MIC ≤ 0.28 µM) and weak to moderate activity towards the fungi C. albicans and C. neoformans. Scheme 1. General method for the synthesis of target polyamine analogues 7-9. Reagents and conditions: (i) for 7a-f: hyodeoxycholic acid (2.0 equiv.), Boc-protected polyamine (6a-f) (1.0 equiv.), PyBOP (2.2 equiv.), DIPEA (6 equiv.), r.t., N 2 , 18 h, (yields 12-96%) or for 8a-f and 9a: carboxylic acid 4 or 5 (2.0 equiv.), Boc-protected polyamine (6a-f) (1.0 equiv.), HOBt (1.0 equiv.), DIPEA (4.0 equiv.), 0 • C, N 2 , 10 min. then EDC·HCl (3.0 equiv.), r.t., N 2 , 18 h (yields 71-92%); (ii) TFA (0.2 mL), CH 2 Cl 2 (2 mL), r.t., 2 h (yields 59-98%).

Cytotoxic and Hemolytic Activities
As noted in a number of studies, amphipathic squalamine mimics can exhibit varying degrees of cytotoxicity and/or hemolytic properties. Cytotoxicity towards HEK293 (human kidney epithelial cell line, IC 50 ) and hemolytic activity against human red blood cells (HC 10 ) were determined for compounds 7-9 ( Table 2). While the HDCA analogues 7a-f were devoid of cytotoxicity and hemolytic properties at the top dose tested (32 µg/mL) and CDCA analogue 9a exhibited low levels of toxicity (IC 50 and HC 10 27 µM), the UDCA analogues 8a-f exhibited hemolytic properties with 8f also exhibiting cytotoxic properties. These results identified the HDCA series of analogues 7a-f as being selective for biological activity towards microorganisms versus mammalian cells. A similar lack of toxicity indicators was also observed for squalamine.

Compound
Cytotoxicity a Hemolysis b

Membrane Perturbation-ATP Release
Previous studies have noted the ability of squalamine to induce rapid loss of intracellular ATP [5,7]. Using the same bioluminescence method, the ability of HDCA analogue 7e to disrupt the membrane of S. aureus was investigated, with the detection of enhanced levels of extracellular ATP used as a reporter reflecting the permeabilizing effect of the compound. CTAB positive control dramatically disrupted the S. aureus membrane after 2 min, leading to observation of pronounced levels of fluorescence ( Figure 6). In direct con-

Membrane Perturbation-ATP Release
Previous studies have noted the ability of squalamine to induce rapid loss of intracellular ATP [5,7]. Using the same bioluminescence method, the ability of HDCA analogue 7e to disrupt the membrane of S. aureus was investigated, with the detection of enhanced levels of extracellular ATP used as a reporter reflecting the permeabilizing effect of the compound. CTAB positive control dramatically disrupted the S. aureus membrane after 2 min, leading to observation of pronounced levels of fluorescence ( Figure 6). In direct contrast, however, was the inability of 7e to induce ATP release, even when examined over a 30 min period after compound exposure. This was a somewhat surprising result, suggesting that the mechanism of anti-Staphylococcus action of 7e does not rely upon targeting the integrity of the bacterial membrane.

Membrane Perturbation-ATP Release
Previous studies have noted the ability of squalamine to induce rapid loss of intracellular ATP [5,7]. Using the same bioluminescence method, the ability of HDCA analogue 7e to disrupt the membrane of S. aureus was investigated, with the detection of enhanced levels of extracellular ATP used as a reporter reflecting the permeabilizing effect of the compound. CTAB positive control dramatically disrupted the S. aureus membrane after 2 min, leading to observation of pronounced levels of fluorescence ( Figure 6). In direct contrast, however, was the inability of 7e to induce ATP release, even when examined over a 30 min period after compound exposure. This was a somewhat surprising result, suggesting that the mechanism of anti-Staphylococcus action of 7e does not rely upon targeting the integrity of the bacterial membrane.

Antibiotic Enhancing Activities
Squalamine and some mimics can enhance the activity of antibiotics towards Gramnegative bacteria [9,21,22]. A sub-set of the HDCA analogues (7a, 7c, 7d, 7e) were tested for the ability to enhance the antibiotic activity of doxycycline against P. aeruginosa ATCC 27853 and of erythromycin against E. coli ATCC 25922. No enhancement was observed for any of the compounds.

General Procedure B: Amide Bond Formation for Cholic Acid Derivatives 8a-f and 9a
To an ice-cold solution of the appropriate cholic acid (2 equiv.) in dry DMF (1 mL) was added Boc-protected polyamine (1 equiv.), HOBt (1 equiv.) and DIPEA (4 equiv.). The solution was stirred for 10 min at 0 • C under N 2 atmosphere. EDC·HCl (3 equiv.) in dry DMF (1 mL) was added to the solution and the resulting mixture was left to stir for 18 h at rt under N 2 atmosphere. The resulting solution was added to EtOAc (20 mL) and washed with H 2 O (2 × 20 mL). The organic layer was dried under reduced pressure and the crude product was purified with silica gel column chromatography (CH 2 Cl 2 /MeOH, 80:20→90:10) to afford the desired Boc-protected product.
[α] 20 solutions of compound 7e as well as 190 µL of a 5 × 10 5 CFU/mL of the selected bacterial suspension in brain heart infusion (BHI) broth. Positive controls containing only 200 µL of a 5 × 10 5 CFU/mL of bacterial suspension in BHI and negative controls containing only 200 µL of BHI broth were added. The plate was incubated at 37 • C in a TECAN Spark Reader (Roche Diagnostic) and real time bacterial growth was followed with OD 590 nm measurements every 10 min during 19 h.

Minimum Bactericidal Concentration Test
A pure culture of a specified microorganism was grown overnight, then diluted in growth-supporting broth (typically Mueller Hinton II broth) to a concentration between 1 × 10 5 and 1 × 10 6 CFU/mL. A stock dilution of the antimicrobial test compound was made at approximately 100 times the expected, previously determined MIC. Further 1:1 dilution was made in 96 well microtiter plates. All dilutions of the test compound were inoculated with equal volumes of the specified microorganism (typically 100 µL). A positive and negative control tube or well was included to demonstrate adequate microbial growth over the course of the incubation period and media sterility, respectively. An aliquot of the positive control was plated and used to establish a baseline concentration of the microorganism used. The microtiter plates were then incubated at 37 • C for 24 h. Turbidity indicates growth of the microorganism, and the MIC is the lowest concentration where no growth was visually observed. To determine the minimum bactericidal concentration (MBC), the dilution representing the MIC and at least two of the more concentrated test product dilutions was plated on a solidified agar plate to determine the bacterial viability. The MBC is the lowest concentration where no growth is encountered when compared to the MIC dilution.

ATP Release Assay
Solutions of the test compound 7e were prepared in DMSO at various concentrations. A suspension of growing S. aureus to be studied in Muller-Hinton II broth was prepared and incubated at 37 • C. An aliquot (90 µL) of this suspension was added to 10 µL of test compound solution and vortexed for 10 s. Luciferin-luciferase reagent (Yelen, France; 50 µL) was immediately added to this mixture, and luminescent signal quantified with an Infinite M200 microplate reader (Tecan) over a 30 min period; ATP concentration was quantified using internal sample addition. A similar procedure was used for the CTAB positive control.

Determination of Antibiotic Enhancement
Restoring enhancer concentrations were determined with an inoculum of 5 × 10 5 CFU in 200 mL of MH broth containing two-fold serial dilutions of each derivative in the presence of either doxycycline or erythromycin at 2 µg/mL. The lowest concentration of the polyamine adjuvant that completely inhibited visible growth after incubation for 18 h at 37 • C was determined. These measurements were independently repeated in triplicate.

Conclusions
In summary, we have synthesized a focused set of dimeric deoxycholic acid-based polyamine derivatives that explored variation in the cholic acid head group (hyodeoxycholic acid, ursodeoxycholic acid and chenodeoxycholic acid) as well as variation in polyamine chain length. Preliminary antimicrobial activities were evaluated against one Gram-positive (S. aureus MRSA), four Gram-negative (E. coli, P. aeruginosa, A. baumannii, K. pneumoniae) bacteria and two fungi (Candida albicans and Cryptococcus neoformans). Many of the compounds exhibited pronounced activity towards S. aureus MRSA with some also exhibiting potent antifungal activity. The overall set of analogues were noticeably inactive against all the target Gram-negative bacteria. Counter-screening for toxicity indicators identified HDCA analogues 7a-f and the sole CDCA analogue 9a to be devoid of cytotoxicity towards the HEK293 and to be non-hemolytic. The observation of cytotoxicity and/or hemolytic activities for the UDCA analogues 8a-f indicates quite a precise structural requirement for bacterial versus mammalian cell toxicity. HDCA analogue 7e exhibited an MBC/MIC ratio of approximately one against three Gram-positive bacteria strains, identifying it to be a bactericidal agent. In a preliminary evaluation of its mechanism of action, 7e failed to cause ATP release from S. aureus cells, a somewhat surprising result given squalamine and many squalamine mimics are reported to target and disrupt bacterial membranes. Together, the present study identifies HDCA-polyamine analogues as being worthy of further study as potent, non-toxic, Gram-positive bactericides with a seemingly unexpected mechanism of action.